An example of a good summary and critical analysis

This was posted by Dr Claudia Stone as a template for us to follow.

Lammich et al. (2004) Expression of the Alzheimer protease BACE1 is suppressed via its 50-untranslated region.

1) Summary:
· Specific questions
o The entire study focused around whether the 5’-untranslated region of BACE1 mRNA was responsible for translational repression of the BACE1 protein, and specifically what characteristics of the 5’UTR are responsible for the observed repression.
Theoretical context
It is assumed that the cause of Alzheimer’s disease is linked to the collection of the amyloid β-peptide (Aβ).
This is created by the actions of two proteases on the membrane amyloid precursor protein.
γ- secretase
β- secretase
Also known as BACE1
Previous studies.
Vassar, 2002
Mice with targeted deletion of BACE1 do not produce any Aβ.
These mice show no any overt phenotype making BACE1 an ideal drug target.
Fukumoto et al, 2002; Holsinger et al, 2002; Yang et al, 2003
BACE1 protein levels are significantly upregulated in the brains of AD patients compared with non-AD controls.
Yasojima et al, 2001; Holsinger et al, 2002; Preece et al, 2003
These increased BACE1 levels corresponded to unchanged mRNA levels.
Suggests that post-transcriptional mechanisms are at play.
Why pinpoint the 5’UTR as the key?
The structure of this segment is:
446 nucleotides long
GC content of 77%
Three uORFs
All of these characteristics are assumed to be important for the inhibition of translation.
Importance of these questions
Determining mechanisms for the regulation of the BACE1 protein, especially at the translational level, would give ideal targets for future therapy in the progression and prevention of Alzheimer’s disease.
Key experiments with results
Determined whether the 5’ UTR may affect the expression of BACE1.
Expression vectors encoding the ORF of BACE1 alone, with the 5’UTR, or with the 3’UTR were transiently transfected into human embryonic kidney HEK293 cells.
Detection done via immunoblotting of the cell lysate.
Showed the presence of the 5’UTR greatly reduced BACE1 protein levels.
Determined whether the BAE1 5’ UTR could inhibit the expression of a similar downstream open reading frame different from BACE1.
Vectors encoding luciferase with or without the 5’UTR, or an empty control vector, were expressed in HEK293 cells.
Luciferase activity was measured in cell lysates.
Luciferase activity was greatly reduced in cells containing the 5’UTR of BACE1.
Proved that the 5’UTR lowered BACE1 protein levels by selectively reducing the translation of BACE1.
Vectors encoding BACE1, with or without the 5’UTR or empty control vector, were expressed in HEK293 cells.
BACE1 protein was measured by immunoblotting cell lysates.
mRNA levels were measured via northern blotting.
The presence of the 5’UTR had no significant effect on mRNA levels, while simultaneously showing lowered BACE1 protein levels.
Additionally demonstrated that the 5’UTR represses the expression of BACE1 at the translational level.
In vitro-transcribed BACE1 mRNA, with and without the 5’UTR, were translated in a nuclease-treated rabbit reticulocyte lysate.
BACE1 protein was detected without the 5’UTR, however was not observed when using the 5’UTR.
Determined whether the high GC content of the long 5’UTR is sufficient for repressing BACE1 expression or whether the uORFs and their encoded short peptides are required
Mutated the start codon of three uORFs from ATG to ATA
Mutated BACE1 plasmids were transfected into HEK293-APP cells.
BACE1 protein levels were measured in the cell lysate by immunoblotting.
Combined mutations of upstream ATGs showed a slight but significant increase in BACE1 expression compared with single mutations
Reveals that the uORFs account for only partial repression of BACE1 expression
Investigated the effect of several deletion mutants of the 5’UTR of BACE 1 with lowered GC content.
Mutations occurred either at nucleotides 1-223, 224-446, or 1-390 and the expression of BACE1 protein was measured.
Showed that both the 5’- and the 3’half of the UTR have a strong inhibitory effect, with it being more pronounced at the 5’ end.

2) Critical Analysis

The article is a systematic progression of the author’s ideas and logic in the development of the study. The reader is given a clear background discussion to serve as the introduction to the topic and why the author has chosen to formulate such a study. Each experiment is explained in the results and discussion section, and reasoning is given unto why the next experiment should be carried out. Additionally, each experiment is explained concisely, with the necessary specifics laid out in the methods section, allowing the reader to follow the thought process of the author and constantly anticipate the next direction of the study.

There is more than enough evidence supporting the author’s claim that the 5’UTR is responsible for repressing the translation of BACE1 without the need to repress transcription. Most of the experiments were designed to test this hypothesis explicitly, and even when it had been proven with an experiment, the study goes one step further by confirming it with an additional experiment.

The study loses the flow of direct evidence during the discussion that the “GC-rich region of the 5’UTR forms a constitutive transition barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA.” The author states that the 5’UTR repression is functioning because of either the high GC content, or because of the uORFs. An experiment is conducted that refutes the idea of the uORFs, however the author immediately states that it must because of the GC content creating a tightly folded secondary structure. Although computer modeling (MFOLD program) of the 5’UTR shows that it’s free energy is sufficient for inhibiting translation, no subsequent testing of this ribosome blocking theory is carried out. An additional experiment is carried out which shows that substituting certain regions of the GC-rich sections of the 5’UTR does indeed increase expression of the BACE1 protein. My belief is that the author is using the previous studies of Wood et al, 1996 and Clemens & Bommer, 1999 as support for the ribosome assumption, but without direct reference.

I agree with the author that these studies are important. The mere fact that there are over 24 million cases of dementia worldwide with around 60% due to AD, shows that identifying a specific mechanism and target for therapy could benefit many individuals. Because the specific, and probably varied, cause of the disease remains undiscovered, the ability to block a mechanism this far downstream would negate may factors that reside earlier, such as at the chromosomal level. Thus a treatment designed at this point could be applied to many patients regardless of disease origin.